诱导多能干细胞自发现以来，以其广阔的再生医学应用前景，受到了极大的关注。到目前为止，已经有大量的工作对其机理进行研究。这其中，表观遗传机制被认为在这一重编程过程中发挥了重要的作用。通过使用转基因手段和小分子化合物改变表观遗传修饰酶的表达水平和活性，科研工作者鉴定了一系列参与调控iPS细胞产生的表观遗传修饰酶，并对相关作用机制做了初步的分析。为了进一步阐明表观遗传修饰酶在特定基因乃至全基因组的特异性定位，与iPS重编程之间的关系，在本项目中，我们拟在不同条件所诱导的体细胞重编程中，表达不同的与dCas9和TALE融合的表观遗传修饰酶，以此为基础，全面研究这些融合蛋白对iPS重编程的影响，并开发出单纯使用这些融合蛋白诱导iPS细胞的新方法。

As its therapeutic potential in regenerative medicine, iPS cell has attracted huge interest since its discovery. So far, the mechanism underlying generation of iPS cells has been thoroughly investigated. Previous studies already demonstrated that epigenetic regulation is pivotal for successful iPS reprogramming. Using genetic and pharmacological approach to perturb expression level or enzymatic activity, dozens of epigenetic enzymes have been identified to be involved in the iPS reprogramming. However, the related mechanisms remain largely unknown. In this proposal, we want to clarify the relationship between iPS reprogramming and the recruitment of epigenetic enzymes to specific chromatin loci nearby certain genes or along whole genome. For this purpose, we will fuse well-designed dCas9 or TALE with various epigenetic enzymes required for generation of iPS cells, and examine their effects under various reprogramming contexts. In addition, we will also try to induce iPS cells from MEFs, using these fusion proteins only.

通过本项目的实施，我们构建了dCas9-VPR、dCas9-P300、dCas9-Tet1CD、dCas9-KRAB、dCas9-LSD1、dCas9-DNMT3α以及dCas9-Sin3α这7个dCas9的融合蛋白，以及靶向小鼠胚胎干细胞超级增强子的sgRNA文库。在结合Drop-seq技术后，我们将可以从中筛选出能够诱导iPS细胞的特异性组合。此外，我们还意外发现了一个能够研究主动DNA去甲基化的系统，并在此基础上筛选出了三个能够促进主动DNA去甲基化的小分子化合物。我们的研究结果不仅为开发诱导iPS细胞的新方法奠定了良好的基础，而且还为研究细胞重编程中的表观遗传机制，提供新的思路与方法。